JP2003515318A - Methods for identifying novel genes involved in the regulation of angiogenesis, testing of such genes and their use for therapeutic purposes - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for identifying a novel gene encoding a cellular component involved in the regulation of angiogenesis, comprising the following steps: a) On an extracellular matrix protein according to four different conditions. Of endothelial cells: standard conditions, conditions for promoting angiogenesis, conditions for inhibiting angiogenesis, control conditions, b) isolation of messenger RNA derived from cells cultured according to various conditions, c) cell components involved in the regulation of angiogenesis For identifying a messenger RNA exclusively or particularly highly induced from cell culture under conditions that stimulate and / or inhibit angiogenesis, corresponding to the gene encoding Qualitative and / or quantitative comparison of different populations of RNA. The invention also relates to a kit for performing the above method.

Description

Detailed Description of the Invention

The present invention is directed to the identification of novel genes encoding cellular components involved in the regulation of angiogenesis. The invention relates in particular to a method allowing the identification of these genes.
The invention also provides for the clinical testing of the factors encoded by the identified genes for the process of angiogenesis, for the diagnosis and treatment of diseases associated with this process, and for the pharmacological, pharmacogenomic or It relates to its use for pharmacological signaling studies.

Angiogenesis is the basic process by which new blood vessels are formed. This process is important in some normal physiological phenomena such as reproduction, development, and even scarring. In these normal biological phenomena, angiogenesis is under tight control, that is, it is initiated during a short period of several days and then completely suppressed. However, some diseases lead to invasive and uncontrolled angiogenesis. For example, arthritis is a disease that results from damage to cartilage by invasive new blood vessels. In diabetic retinopathy, invasion of the retina by neovascularization leads to blindness in patients, that is, neovascularization of the ocular device is a major cause of blindness, and this neovascularization controls 20 eye diseases. There is. Finally, tumor growth and metastasis are directly linked to neovascularization and are dependent on angiogenesis. Tumors stimulate the growth of new blood vessels for their growth. In addition, these new blood vessels provide a leaky pathway for tumors to connect to the blood circulation and cause metastases at distant sites such as the liver, lungs or bones.

In other diseases such as cardiovascular disease, peripheral arterial disease, vascular or brain lesions,
Angiogenesis can provide an important therapeutic platform. In fact, the promotion of angiogenesis in the injured area is a lateral alternative to the injured blood vessels that supplies blood and, consequently, oxygen and other nutrients and biological factors required for survival of the tissues involved. It can lead to the formation of new blood vessels.

The formation of new blood vessels by endothelial cells involves the migration, proliferation and differentiation of endothelial cells. Regulation of these biological phenomena is directly linked to gene expression. With respect to angiogenesis, an ever-increasing number of tests have revealed that angiogenesis is conducted through a balance between factors that act directly on endothelial cells. These factors are believed to be stimulators such as VEGF, FGF, IL-8, HGF / SF, PDGF, etc., on the one hand. On the other hand, it may be an inhibitor such as IL-10, IL-12, glo-α and β, platelet factor 4, angiostatin, human chondrocyte-derived inhibitor, thrombospondin, leukemia inhibitor, etc. (Jensen
, Surg. Neural. , 1998, 49, 189-195; Tamata.
ni et al., Carcinogenesis, 1999, 20, 957-962; T.
anaka et al., Cancer Res. , 1998, 58, 3362-3369
Ghe et al., Cancer Res. , 1997, 57, 3733-3740;
Kawahara et al., Hepatology, 1998, 28, 1512-15.
17; Chanduni et al., Cancer Res. , 1997, 57, 18
14-1819; Jendraschak and Sage, Semin. Cancel
r Biol. , 1996, 7, 139-146; Majewski et al. I
nvest. Dermatol. , 1996, 106, 1114-1119).

Regulation of angiogenesis, therefore, is not only one of the strategic backbones of basic research to enhance our understanding of the many pathological phenomena linked to angiogenesis, but also to treat diseases associated with angiogenesis. It is also the basis for the development of new treatments intended to do so.

To control angiogenesis, several pharmaceutical groups have developed therapeutic strategies directly based on the use of paracrine signals, stimulators and inhibitors, as agents to promote or inhibit angiogenesis. did. These strategies basically use these factors in protein form as stimulators and inhibitors of angiogenesis,
Alternatively, more recently it is based on use in the form of an expression vector encoding the factor of choice.

The availability of test models that allow experiments to be conducted in vitro will facilitate the discovery of novel molecules that can be used as active ingredients useful in the treatment of diseases associated with angiogenesis.

Endothelial cells can be cultured. The first method of culturing consists of attaching the cells to the flat bottom of a culture flask, immersing them in medium and then incubating until a confluent cell layer is obtained. Another method of culturing is characterized by attaching endothelial cells to the flat bottom of a culture flask which has been previously coated with a layer of proteins belonging to the protein family called extracellular matrix proteins. In yet another method, endothelial cells are coated with a lattice obtained by retractant proteins of this family, such as collagen or fibrin, which form collagen and fibrin lattices, respectively.

In culturing endothelial cells, regardless of the technique used, a uniform monolayer of cell population is obtained after incubation for a certain period of time. Nevertheless, each population thus obtained has a structure independent of the mechanism of the differentiated endothelial cells forming new blood vessels.

It has already been shown that endothelial cell cultures under collagen or fibrin lattices result in differentiated endodermal cells forming capillaries after stimulation of the endothelial cell layer with one of the paracrine signals that promote angiogenesis. (Montesano et al., Proc. Natl. Acad. Sci. SUA, 1986, 83, 7297).
-7301). These capillaries formed in vitro by stimulated endothelial cells resemble new blood vessels formed by the same cell type in vivo during angiogenesis. Therefore, they constitute a suitable model for the study of new factors involved in the regulation of angiogenesis.

Regulation of angiogenesis occurs through a balance between the actions of stimulators and inhibitors. These factors, which are themselves products of gene expression, are mitogenic. They regulate the expression of several genes that are involved in the regulation of angiogenesis and may also be involved in other physiological or pathological processes. Two gene families are involved in the regulation of angiogenesis. One is a first generation gene family that encodes stimulators or inhibitors. On the other hand, regulation of angiogenesis such as cell receptors, extracellular matrix proteins, docking proteins (“docking proteins”), cross-linking proteins (“adaptor proteins”), kinases, phosphatases, proteases, glycosyltransferases, etc. It is a gene family that encodes cell components closely related to. This gene family therefore constitutes the "second generation gene family of angiogenesis".

The present invention relates to a method for identifying a gene that encodes a cellular component closely related to regulation of angiogenesis and belongs to the second generation gene family of angiogenesis. What is a cell component?
It means a protein, protein group or set of proteins of the type defined below for the products encoded by the genes of the second generation family. Factors that stimulate or inhibit this process control the expression of genes involved in increasing or suppressing angiogenesis.
Therefore, two gene expression profiles have to be considered. The first corresponds to the profile of cells placed under conditions for promoting angiogenesis and the second corresponds to cells placed under conditions for inhibiting angiogenesis.

Although the physiological states of unstimulated endothelial cells and cells under conditions of inhibiting angiogenesis are similar, it is important to distinguish gene expression in the two cases. Indeed, endothelial cells stimulated with factors that promote angiogenesis, such as FGF1, and incubated with angiogenesis inhibitors are unable to form new blood vessels. In addition, some factors that inhibit angiogenesis are mitogens and IL
-10, IL-12, glo-α and β, etc., affect gene expression. Thus, endothelial cells may exhibit gene expression under angiogenesis-inhibiting conditions that differ from those of unstimulated endothelial cells. Therefore, it is important to compare the gene expression profile of cells under different culture conditions, i.e. angiogenic and inhibitory conditions, with well defined standard culture conditions. Is.

The method according to the invention makes it possible to identify genes which code for cellular components involved in the regulation of angiogenesis. The method comprises the following steps: a) Culturing of endothelial cells on extracellular matrix proteins according to four different conditions: standard conditions, angiogenesis promoting conditions, angiogenesis inhibiting conditions, control conditions, b) Isolation of messenger RNA derived from cells cultured according to various conditions, c) under conditions that stimulate and / or inhibit angiogenesis, corresponding to genes encoding cellular components involved in the regulation of angiogenesis. Qualitative and / or quantitative comparison of different populations of messenger RNAs to identify messenger RNAs that are exclusively or particularly highly induced from cell cultures, d) optionally a step ( isolation of the messenger RNAs identified in c), their amplification and purification, e) optionally cloning and sequence of the nucleic acid molecule obtained in the above step F) optionally, one or more genes corresponding to the isolated nucleic acid molecules, including expression of said genes in a suitable system and testing of the protein so produced for properties in regulating angiogenesis. Identification.

To identify members of the second generation family of genes involved in the regulation of angiogenesis,
The inventor has determined four experimental conditions for endothelial cell culture: Cells cultivated under standard conditions (CR) are not stimulated. -Cells cultured under pro-angiogenic conditions (CPA) are stimulated by angiogenic factors. Angiogenic factors can be, for example, FGF1, FGF2, HGF, PDGF, and so on. Cells cultivated under angiogenesis-inhibiting conditions (CIA) are stimulated by angiogenic factors and incubated with one or more anti-angiogenic factors. The stimulator is FGF2 and the inhibitor may be selected from, for example, IL-10, IL-12, the chemokine glo-α or β, and the like. -Cells cultured under control conditions (CC) are incubated with anti-angiogenic factors. Anti-angiogenic factors include, for example, IL-10, IL-12, the chemokine glo-alpha
Or β, and so on.

These experimental culture conditions apply to endothelial cells cultured according to any of the methods known to those skilled in the art. Extracellular protein substrates include fibrin, collagen,
It may be constituted by laminin, Matrigel, fibronectin or any other protein belonging to the extracellular matrix protein family.

The factors capable of stimulating or inhibiting angiogenesis are selected from among the factors whose effects on the process of angiogenesis are known (Jensen, Su.
rg. Neural. , 1998, 49, 189-195; Tamatani et al., Carcinogenesis, 1999, 20, 957-962; Tana.
ka et al., Cancer Res. , 1998, 58, 3362-3369; Gh
e et al., Cancer Res. , 1997, 57, 3733-3740; Kaw
ahara et al., Hepatology, 1998, 28, 1512-1517;
Chanduni et al., Cancer Res. , 1997, 57, 1814-
1819; Jendraschak and Sage, Semin. Cancer B
iol. , 1996, 7, 139-146; Majewski et al. Inve
st. Dermatol. , 1996, 106, 1114-1119).

The angiogenic stimulator used in step (a) of the method according to the invention is selected from: -Fibroblast growth factor 1-15 (FGF1-FGF15) -Epidermal growth factor (EGF) -Vascular Endothelial Growth Factor (VEGF) -Hepatocyte Growth Factor (HGF) -Platelet Derived Growth Factor (PDGF) -Interleukin 8 (IL-8) -Angiogenin-Transforming Growth Factor (TGF) -Neurokine Neurokine midkine-Pleiotropin or some other proteinaceous factor that induces angiogenesis.

The angiogenesis inhibitor used in step (a) of the method according to the invention is selected from: -thrombospondin-angiostatin-endostatin-platelet factor 4-interleukin-10. (IL-10) -Interleukin 12 (IL-12) -Kimiokinglo-α and β-Human chondrocyte-derived inhibitor-Leukemia inhibitor-Tumor necrosis factor (TNF) or some other proteinaceous inhibitor of angiogenesis factor.

Various factors that regulate angiogenesis are added to cell culture according to methods well known to those skilled in the art (Montesano et al., Proc. Natl. Acad. Sci. USA,
1986, 83, 7297-7301). Effective concentrations to be used for each of the various factors are advantageously: 1 ng / ml-200 ng / ml.

In the method according to the invention, one or more factors capable of stimulating or inhibiting angiogenesis are constructed so as to allow the synthesis of the one or more factors in cell culture. Added to the cells in culture in the form of the expressed expression vector. Examples of such vectors are described by Tanaka et al. (Cancer Res., 1998, 58,
3362-3369).

The method according to the invention comprises in step (c) the analysis of the gene expression profile of the cells under conditions of stimulating or inhibiting angiogenesis. This analysis is based on testing endothelial cell messenger RNA under angiogenic stimulatory or inhibitory conditions compared to cellular messenger RNA under standard and control conditions. These messenger RNAs are extracted from cells by classical techniques that can separate RNA from other cellular components (Vancopoulos et al., 1990, "Methods for cloning and analyzing eukaryotic genes (Methods for clones.
ing and analysis of eucarrotic genes
) ”, P. 8-23, edited by Jones and Bartlett, Boston)
. Messenger RNAs are separated from other RNAs, for example by utilizing the presence of poly-A sequences at their ends.

Analysis of different populations of messenger RNA can be carried out by a differential display method comprising the steps of: -reverse transcribing messenger RNA-resolving DNA molecules obtained by reverse transcription by electrophoresis -Detect the DNA molecule of interest and then excise it from the electrophoretic support-purify the DNA of interest-the purified DNA can be used as a probe to screen a cDNA library, or directly It can also be subcloned and sequenced.

The purified DNA can be used as a probe, in particular for screening cDNA libraries or for detecting complementary nucleic acid sequences by Northern blotting.

The differential display method involves simultaneous detection of two groups of differentially expressed genes, eg some genes associated with angiogenic factors selected from (a) and (b). Allows simultaneous detection of another gene associated with an anti-angiogenic factor selected from The differential display technique is also a rare mR
NA can be detected and duplicates and false positive clones can be minimized.

In the method according to the invention, analysis of different populations of messenger RNA can also be carried out by subtractive hybridization.

Subtraction hybridization includes separation of mRNA, reverse transcription of mRNA, construction of a cDNA library, subtraction, and screening by differential hybridization.

These two methods are complementary and are used to identify mRNAs corresponding to angiogenesis.

The method according to the invention is therefore outstanding in that it allows a qualitative and quantitative assessment of angiogenesis under the action of two paracrine signals, angiogenic and antiangiogenic. is doing.

Moreover, this method can test the action of various proteins (fibrin, collagen, laminin, matrigel, fibronectin, etc.) as extracellular matrix in combination with paracrine signals. Moreover, such methods can be readily modified to include other cell types in addition to endothelial cells.

Another aspect of the invention is the diagnosis and / or diagnosis of diseases associated with angiogenesis with factors identified using the method, such as arthritis, diabetic retinopathy, cancer or cardiovascular disease. Or in a pharmaceutical composition intended for treatment. The agents identified using the method according to the invention can also be used for pharmacological, pharmacogenomic or pharmacosignaling studies.

A complementary aspect of the invention consists of a test kit for carrying out the method which is the subject of the present invention. Such kits include materials and reagents useful for culturing endothelial cells on extracellular matrix proteins in combination with one or more angiogenic or anti-angiogenic factors. Advantageously, in the case of test kits intended for diagnosis, one or more of angiogenic factors and anti-angiogenic factors can be replaced by the patient's serum.

Other advantages and features of the invention will become apparent on reading the examples below, given by way of non-limiting example with reference to FIG.

Example 1 Differential Display: FIG. 1 schematically shows a method of differential display of genes involved in angiogenesis.

The mRNA can be oligo (dT) primer, T12MN, where M can be G, A or C; N can be G, A, T or C] each of the four degenerate groups. Used specifically to extract and reverse transcribe.

Each group of primers is defined by a base at the 3 ′ (N) terminus with a degeneracy at the (M) position. For example, a group of primers where N = G is constituted by: 5'-TTTTTTTTTTTTGG-3 '5'-TTTTTTTTTTTTTAG-3'5'-TTTTTTTTTTTTCG-3'.

The gel diagram schematizes the use results of only the first generation group for endothelial cells under the three experimental conditions: CR = standard conditions (cultured endothelial cells without stimulation), CPA = Angiogenesis promoting conditions (endothelial cells stimulated by an angiogenic factor selected from (a) such as FGF2), CIA = angiogenesis inhibiting conditions ((
a) endothelial cells co-stimulated with angiogenic factors from a) and anti-angiogenic factors from (b)), CC = control conditions (endothelial cells stimulated with anti-angiogenic factors selected from among (b)).

The dotted line in FIG. 1 represents RNA and the solid line represents DNA. T12MN is oligo (
dT) degenerate primer; M can be A, G or G (degenerate position); N
Can be A, C, G or T.

Example 2 Subtraction Hybridization FIG. 2 is a schematic representation of the method of subtraction hybridization of cDNA.

MRNA is extracted from the cells and reverse transcribed to obtain single-stranded cDNA representing the sequence of RNA expressed in cells subjected to various operating conditions.

Single-chain cD derived from cells placed under certain operating conditions, such as angiogenic stimulation conditions
NA is an excess of RNA derived from cells that have been subjected to other operating conditions, such as standard conditions.
Hybridize with. In such a hybridization reaction, the cDNA corresponding to the sequences expressed under the two operating conditions will hybridize with RNA. Conversely, cDNA not represented in RNA remains in the form of single-stranded cDNA.

Conveniently, by limiting the amount of mRNA used in the hybridization reaction, the method also allows one to be overexpressed in cells placed under given operating conditions, eg, angiogenic stimulation conditions. Or c representing more mRNA
It can also be used to separate DNA sequences.

Double-stranded cDNA and single-stranded cDNA can be separated by chromatography, especially on a hydroxyapatite column. The single-stranded cDNA thus separated corresponds to the mRNA which is specifically present or overexpressed in cells placed under specific culture conditions. This cDNA identifies the corresponding gene,
It can be used to screen a cDNA library for isolation and cloning.

[Brief description of drawings]

FIG. 1 schematically shows a method of differential display of genes involved in angiogenesis.

FIG. 2 is a diagrammatic representation of a method for subtraction hybridization of cDNA.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] October 8, 2001 (2001.10.8)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

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Claims (12)

[Claims] 1. A method for identifying a gene encoding a cellular component involved in the regulation of angiogenesis, comprising the steps of: a) culturing an endothelial cell on an extracellular matrix protein according to the following four different conditions: -Standard conditions-Conditions that promote angiogenesis-Conditions that inhibit angiogenesis-Control conditions b) A step of separating messenger RNA derived from cells cultured according to various conditions, and c) Control of angiogenesis In order to identify messenger RNAs which are exclusively derived from cell cultures under conditions which stimulate and / or inhibit angiogenesis, corresponding to genes encoding the cellular components involved, or which are induced in particular in high amounts, Comprising the steps of qualitatively and / or quantitatively comparing different populations of messenger RNA, the cellular development involved in the regulation of angiogenesis. Methods for identifying genes encoding. 2. A step of separating the messenger RNAs identified in step (c), amplifying and purifying them, a step of cloning and sequencing the nucleic acid molecule obtained in the above step, Identifying one or more genes corresponding to the separated nucleic acid molecules. 3. Cells cultivated under standard conditions (CR) are not stimulated; cells cultivated under pro-angiogenic conditions (CPA) are stimulated by angiogenic factors; -under angiogenesis-inhibiting conditions (CIA) Characterized in that the cells to be cultured are stimulated by angiogenic factors and incubated with one or more anti-angiogenic factors, -the cells to be cultured under control conditions (CC) are incubated with anti-angiogenic factors The method according to claim 1 or 2. 4. The extracellular matrix according to any one of claims 1 to 3, wherein the extracellular matrix is fibrin, collagen, laminin, Matrigel, fibronectin or any other protein belonging to the extracellular matrix protein family. The method described. 5. Angiogenesis promoting factor used in step (a) is: -Fibroblast growth factor 1-15 (FGF1-FGF15) -Epidermal cell growth factor (EGF) -Vascular endothelial cell growth factor (VEGF)- Hepatocyte growth factor (HGF) -Platelet-derived growth factor (PDGF) -Interleukin 8 (IL-8) -Angiogenin-Transforming growth factor (TGF) -Neurokine midkine-Pleiotropin or angiogenesis 5. The method according to any one of claims 1 to 4, wherein the method is selected from some other proteinaceous factor that induces. 6. The angiogenesis inhibitor used in step (a) of the method of the present invention is: -Thrombospondin-Angiostatin-Endostatin-Platelet factor 4-Interleukin 10 (IL-10) -Interleukin 12 (IL-12) -Kimiokinglo-α and β-Human chondrocyte-derived inhibitor-Leukemia inhibitor-Tumor necrosis factor (TNF) or some other proteinaceous factor that inhibits angiogenesis The method according to claim 1, wherein 7. A form of an expression vector constructed to allow the synthesis of one or more factors in cell culture, wherein the one or more factors capable of stimulating or inhibiting angiogenesis. Characterized in that it is added to cells in culture with
The method according to any one of claims 1 to 6. 8. Analysis of various populations of messenger RNA, the steps of reverse transcribing messenger RNA, separating the DNA molecule obtained by reverse transcription by electrophoresis, and the DNA molecule of interest. And then excising it from the support for electrophoresis, -purifying the target DNA, -using the purified DNA as a probe in some cases, -substituting the purified DNA in some cases by subcloning And a differential display method including a step of determining,
The method according to any one of claims 1 to 7. 9. The method according to claim 1, characterized in that the analysis of different populations of messenger RNA is carried out by subtraction hybridization. 10. Use of a gene identified using the method according to any one of claims 1 to 9 in a pharmaceutical composition intended for the diagnosis and / or treatment of diseases associated with angiogenesis. 11. A test kit intended for the diagnosis and / or treatment of diseases associated with angiogenesis, which comprises one or more test kits capable of carrying out the method according to any one of claims 1 to 9. A kit comprising a substance and a reagent useful for culturing endothelial cells on an extracellular matrix protein in combination with an angiogenic factor or an anti-angiogenic factor. 12. The method according to claim 11, wherein one or more of angiogenic factors and anti-angiogenic factors can be replaced by the serum of the patient.
A test kit intended for the diagnosis described in.